#Setup the working environment
setwd("/hpc/group/pbenfeylab/mz187/")
# Load the basic package_Seurat
suppressMessages(library(tidyverse))
suppressMessages(library(Seurat))
suppressMessages(library(cowplot))
suppressMessages(library(ComplexHeatmap))
suppressMessages(library(circlize))
suppressMessages(library(GeneOverlap))
suppressMessages(library(gprofiler2))
suppressMessages(library(ggrepel))
suppressMessages(library(muscat))
suppressMessages(library(limma))
suppressMessages(library(scran))
suppressMessages(library(future))
suppressMessages(library(RColorBrewer))
Warning message: “package ‘tidyverse’ was built under R version 4.2.3” Warning message: “package ‘tibble’ was built under R version 4.2.3” Warning message: “package ‘tidyr’ was built under R version 4.2.3” Warning message: “package ‘readr’ was built under R version 4.2.3” Warning message: “package ‘purrr’ was built under R version 4.2.3” Warning message: “package ‘dplyr’ was built under R version 4.2.3” Warning message: “package ‘stringr’ was built under R version 4.2.3” Warning message: “package ‘forcats’ was built under R version 4.2.3” Warning message: “package ‘lubridate’ was built under R version 4.2.3” Warning message: “package ‘ComplexHeatmap’ was built under R version 4.2.1” Warning message: “package ‘circlize’ was built under R version 4.2.3” Warning message: “package ‘GeneOverlap’ was built under R version 4.2.1” Warning message: “package ‘gprofiler2’ was built under R version 4.2.3” Warning message: “package ‘muscat’ was built under R version 4.2.2” Warning message: “package ‘limma’ was built under R version 4.2.3” Warning message: “package ‘scran’ was built under R version 4.2.3” Warning message: “package ‘SingleCellExperiment’ was built under R version 4.2.2” Warning message: “package ‘SummarizedExperiment’ was built under R version 4.2.2” Warning message: “package ‘MatrixGenerics’ was built under R version 4.2.1” Warning message: “package ‘matrixStats’ was built under R version 4.2.3” Warning message: “package ‘GenomicRanges’ was built under R version 4.2.3” Warning message: “package ‘BiocGenerics’ was built under R version 4.2.1” Warning message: “package ‘S4Vectors’ was built under R version 4.2.3” Warning message: “package ‘IRanges’ was built under R version 4.2.3” Warning message: “package ‘GenomeInfoDb’ was built under R version 4.2.3” Warning message: “package ‘Biobase’ was built under R version 4.2.3” Warning message: “package ‘scuttle’ was built under R version 4.2.3” Warning message: “package ‘RColorBrewer’ was built under R version 4.2.3”
# Read integrated Copilot project
rc.integrated <- readRDS("Rice_8S_snRNA-seq_seu3_filtered-102824.rds")
# Find included samples
table(rc.integrated$orig.ident)
sc_295 sc_296 sc_297 sc_298 sc_299 sc_300 sc_301 sc_302 2314 4264 9173 7046 6926 6912 9593 8797
# Check the seurat clusters for the integrated object
options(repr.plot.width=16, repr.plot.height=16)
DimPlot(rc.integrated, reduction = "umap", group.by = "seurat_clusters", label = T)
Warning message:
“Using `as.character()` on a quosure is deprecated as of rlang 0.3.0. Please use `as_label()` or `as_name()` instead.
This warning is displayed once every 8 hours.”
# Assign cell type annotation to the clusters
rc.integrated$manual.anno <- as.character(rc.integrated$seurat_clusters)
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='0')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='1')] <- "Exodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='2')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='3')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='4')] <- "Exodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='5')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='6')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='7')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='8')] <- "Cortex"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='9')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='10')] <- "Mature root cells"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='11')] <- "Trichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='12')] <- "Xylem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='13')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='14')] <- "Xylem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='15')] <- "Exodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='16')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='17')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='18')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='19')] <- "Atrichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='20')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='21')] <- "Sclerenchyma"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='22')] <- "Sclerenchyma"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='23')] <- "Atrichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='24')] <- "Phloem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='25')] <- "Atrichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='26')] <- "Exodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='27')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='28')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='29')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='30')] <- "Mature root cells"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='31')] <- "Xylem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='32')] <- "Trichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='33')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='34')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='35')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='36')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='37')] <- "Phloem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='38')] <- "Atrichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='39')] <- "Endodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='40')] <- "Trichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='41')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='42')] <- "Endodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='43')] <- "Phloem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='44')] <- "Trichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='45')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='46')] <- "Sclerenchyma"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='47')] <- "Exodermis"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='48')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='49')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='50')] <- "Pericycle"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='51')] <- "Meristem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='52')] <- "Trichoblast"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='53')] <- "Xylem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='54')] <- "Phloem"
rc.integrated$manual.anno[which(rc.integrated$seurat_clusters=='55')] <- "Phloem"
table (rc.integrated$manual.anno)
Atrichoblast Cortex Endodermis Exodermis
4022 1520 1255 6392
Mature root cells Meristem Pericycle Phloem
2294 17739 8669 2662
Sclerenchyma Trichoblast Xylem
2808 3804 3860
# Make final cell type annotation figure
order <- c("Meristem", "Atrichoblast", "Trichoblast", "Exodermis","Sclerenchyma","Cortex", "Endodermis","Pericycle", "Phloem", "Xylem","Mature root cells")
palette <- c("#360167","#59adf6", "#Ff4061", "#F8f38d", "#32CD32", "#BF40BF", "#Ffb480","#08cad1", "#9d94ff","#834200","#b7bcc0")
rc.integrated$manual.anno <- factor(rc.integrated$manual.anno, levels = order[sort(match(unique(rc.integrated$manual.anno),order))])
color <- palette[sort(match(unique(rc.integrated$manual.anno),order))]
options(repr.plot.width=12, repr.plot.height=12)
DimPlot(rc.integrated, reduction = "umap", group.by = "manual.anno", cols= palette, pt.size = 0.1)+ggtitle("Celltype Annotation UMAP")
## Subgroup the "normal" groups for the following analysis
rc.kitaake <- subset(rc.integrated, cells=colnames(rc.integrated)[which(rc.integrated$condition!="X.K")])
table(rc.kitaake$gel_soil)
non-compact soil compact soil gel
13838 18390 16219
table(rc.kitaake$gel_soil, rc.kitaake$orig.ident)
sc_297 sc_298 sc_299 sc_300 sc_301 sc_302
non-compact soil 0 0 6926 6912 0 0
compact soil 0 0 0 0 9593 8797
gel 9173 7046 0 0 0 0
## Subgroup the "normal" groups for the following analysis
rc.comparison <- subset(rc.kitaake, cells=colnames(rc.kitaake)[which(rc.kitaake$gel_soil!="non-compact soil")])
# Define the datasets used for Gel VS Soil comparison
rc.comparison$normal <- rc.comparison$orig.ident %in% c("sc_297","sc_298")
rc.comparison$normal <- as.character(rc.comparison$normal)
rc.comparison$normal <- gsub("TRUE", "gel", rc.comparison$normal,
ignore.case = FALSE, perl = FALSE,
fixed = T, useBytes = FALSE)
rc.comparison$normal <- gsub("FALSE", "soil", rc.comparison$normal,
ignore.case = FALSE, perl = FALSE,
fixed = T, useBytes = FALSE)
rc.comparison$normal <- factor(rc.comparison$normal ,
levels=c("gel", "soil"))
table(rc.comparison$normal, rc.comparison$orig.ident)
sc_297 sc_298 sc_301 sc_302
gel 9173 7046 0 0
soil 0 0 9593 8797
table(rc.comparison$normal, rc.comparison$manual.anno)
Meristem Atrichoblast Trichoblast Exodermis Sclerenchyma Cortex
gel 8540 624 646 1701 788 171
soil 3148 2218 1505 2051 936 776
Endodermis Pericycle Phloem Xylem Mature root cells
gel 381 2075 142 1076 75
soil 238 3316 1212 1936 1054
# construct sce manually
my_metadata <- data.frame(sample_id = rc.comparison$orig.ident,
group_id = rc.comparison$normal,
cluster_id = rc.comparison$manual.anno)
head(my_metadata)
sce <- SingleCellExperiment(assays = list(counts = rc.comparison@assays$RNA@counts),
colData = my_metadata)
sce
(sce <- prepSCE(sce,
kid = "cluster_id", # subpopulation assignments
gid = "group_id", # group IDs (ctrl/stim)
sid = "sample_id", # sample IDs (ctrl/stim.1234)
drop = TRUE)) # drop all other colData columns
| sample_id | group_id | cluster_id | |
|---|---|---|---|
| <chr> | <fct> | <fct> | |
| AAACCCAAGATGAATC_1 | sc_297 | gel | Exodermis |
| AAACCCAAGGACGCTA_1 | sc_297 | gel | Pericycle |
| AAACCCAAGGATATGT_1 | sc_297 | gel | Atrichoblast |
| AAACCCAAGGCACCAA_1 | sc_297 | gel | Cortex |
| AAACCCAAGTCCCTAA_1 | sc_297 | gel | Exodermis |
| AAACCCACAACCCGCA_1 | sc_297 | gel | Meristem |
class: SingleCellExperiment dim: 41204 34609 metadata(0): assays(1): counts rownames(41204): LOC-Os01g01010 LOC-Os01g01019 ... LOC-Os09g27120 LOC-Os09g39170 rowData names(0): colnames(34609): AAACCCAAGATGAATC_1 AAACCCAAGGACGCTA_1 ... TTTGTTGTCTCGGTCT_8 TTTGTTGTCTGATTCT_8 colData names(3): sample_id group_id cluster_id reducedDimNames(0): mainExpName: NULL altExpNames(0):
class: SingleCellExperiment dim: 41204 34609 metadata(1): experiment_info assays(1): counts rownames(41204): LOC-Os01g01010 LOC-Os01g01019 ... LOC-Os09g27120 LOC-Os09g39170 rowData names(0): colnames(34609): AAACCCAAGATGAATC_1 AAACCCAAGGACGCTA_1 ... TTTGTTGTCTCGGTCT_8 TTTGTTGTCTGATTCT_8 colData names(3): cluster_id sample_id group_id reducedDimNames(0): mainExpName: NULL altExpNames(0):
# Store cluster and sample IDs, as well as the number of clusters
# and samples into the following simple variables
nk <- length(kids <- levels(sce$cluster_id))
ns <- length(sids <- levels(sce$sample_id))
names(kids) <- kids
names(sids) <- sids
# Data overview
# nb. of cells per cluster-sample
t(table(sce$cluster_id, sce$sample_id))
Meristem Atrichoblast Trichoblast Exodermis Sclerenchyma Cortex
sc_297 5242 415 298 796 380 71
sc_298 3298 209 348 905 408 100
sc_301 1929 1079 676 787 375 423
sc_302 1219 1139 829 1264 561 353
Endodermis Pericycle Phloem Xylem Mature root cells
sc_297 157 1032 84 660 38
sc_298 224 1043 58 416 37
sc_301 147 1745 670 1069 693
sc_302 91 1571 542 867 361
# Aggregation of single-cell to pseudo-bulk data
pb <- aggregateData(sce,
assay = "counts", fun = "sum",
by = c("cluster_id", "sample_id"))
# one sheet per sub-population
assayNames(pb)
# pseudo-bulks for 1st sub-population
t(head(assay(pb)))
| LOC-Os01g01010 | LOC-Os01g01019 | LOC-Os01g01030 | LOC-Os01g01040 | LOC-Os01g01050 | LOC-Os01g01060 | |
|---|---|---|---|---|---|---|
| sc_297 | 367 | 10 | 168 | 691 | 183 | 1630 |
| sc_298 | 286 | 6 | 141 | 484 | 118 | 1484 |
| sc_301 | 3 | 1 | 2 | 8 | 2 | 32 |
| sc_302 | 3 | 0 | 1 | 12 | 2 | 37 |
# Pseudo-bulk-level MDS plot
(pb_mds <- pbMDS(pb))
Removing 9400 rows with all zero counts
# Sample-level analysis: Pseudo-bulk methods
# construct design & contrast matrix
ei <- metadata(sce)$experiment_info
ei
mm <- model.matrix(~ 0 + ei$group_id)
mm
dimnames(mm) <- list(ei$sample_id, c(levels(ei$group_id)))
mm
contrast <- makeContrasts("gel-soil", levels = mm)
contrast
| sample_id | group_id | n_cells |
|---|---|---|
| <fct> | <fct> | <dbl> |
| sc_297 | gel | 9173 |
| sc_298 | gel | 7046 |
| sc_301 | soil | 9593 |
| sc_302 | soil | 8797 |
| ei$group_idgel | ei$group_idsoil | |
|---|---|---|
| 1 | 1 | 0 |
| 2 | 1 | 0 |
| 3 | 0 | 1 |
| 4 | 0 | 1 |
| gel | soil | |
|---|---|---|
| sc_297 | 1 | 0 |
| sc_298 | 1 | 0 |
| sc_301 | 0 | 1 |
| sc_302 | 0 | 1 |
| gel-soil | |
|---|---|
| gel | 1 |
| soil | -1 |
# run DS analysis
res <- pbDS(pb, design = mm, contrast = contrast,filter = "none")
|======================================================================| 100%
# access results table for 1st comparison
tbl <- res$table[[1]]
# one data.frame per cluster
names(tbl)
# filter FDR < 5%, abs(logFC) > log2(1.5) & sort by adj. p-value # keep 1.5 or not
tbl_fil <- lapply(tbl, function(u) {
u <- dplyr::filter(u, p_adj.loc < 0.05, abs(logFC) > log2(1.5), logCPM >=1)
dplyr::arrange(u, p_adj.loc)
})
# nb. of DS genes & % of total by cluster
n_de <- vapply(tbl_fil, nrow, numeric(1))
p_de <- format(n_de / nrow(sce) * 100, digits = 3)
data.frame("#DS" = n_de, "%DS" = p_de, check.names = FALSE)
| #DS | %DS | |
|---|---|---|
| <dbl> | <chr> | |
| Meristem | 2525 | 6.128 |
| Atrichoblast | 1578 | 3.830 |
| Trichoblast | 1440 | 3.495 |
| Exodermis | 1702 | 4.131 |
| Sclerenchyma | 720 | 1.747 |
| Cortex | 440 | 1.068 |
| Endodermis | 237 | 0.575 |
| Pericycle | 2848 | 6.912 |
| Phloem | 434 | 1.053 |
| Xylem | 1220 | 2.961 |
| Mature root cells | 530 | 1.286 |
# Merge 9 data frames for each single cell type into one big data frame for the future analysis
tb2 <- resDS(sce, res, bind = "row", cpm=FALSE, frq=TRUE)
head(tb2, n = 7)
| gene | cluster_id | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> | |
| 1 | LOC-Os01g01010 | Meristem | 0.05990 | 0.06700 | 0.001560 | 0.00246 | 0.06260 | 0.001910 | 1.3300 | 3.44 | 5.890 | 0.01520 | 0.1140 | 0.158 | gel-soil |
| 2 | LOC-Os01g01019 | Meristem | 0.00191 | 0.00182 | 0.000518 | 0.00000 | 0.00187 | 0.000318 | -1.4100 | -1.26 | 0.522 | 0.47000 | 0.8660 | 0.849 | gel-soil |
| 3 | LOC-Os01g01030 | Meristem | 0.03000 | 0.03730 | 0.001040 | 0.00082 | 0.03280 | 0.000953 | 1.2500 | 2.40 | 2.690 | 0.10100 | 0.3830 | 0.454 | gel-soil |
| 4 | LOC-Os01g01040 | Meristem | 0.11600 | 0.12400 | 0.004150 | 0.00984 | 0.11900 | 0.006350 | 0.4320 | 4.29 | 1.610 | 0.20400 | 0.5750 | 0.628 | gel-soil |
| 5 | LOC-Os01g01050 | Meristem | 0.03190 | 0.03180 | 0.001040 | 0.00164 | 0.03190 | 0.001270 | 0.7880 | 2.37 | 1.260 | 0.26100 | 0.6550 | 0.694 | gel-soil |
| 6 | LOC-Os01g01060 | Meristem | 0.22300 | 0.28300 | 0.016100 | 0.02630 | 0.24600 | 0.020000 | 0.0679 | 5.74 | 0.122 | 0.72700 | 0.9300 | 0.915 | gel-soil |
| 7 | LOC-Os01g01070 | Meristem | 0.07060 | 0.08700 | 0.000518 | 0.00410 | 0.07690 | 0.001910 | 1.5200 | 3.62 | 8.100 | 0.00444 | 0.0472 | 0.070 | gel-soil |
# Save all the gene expression information by Muscat
write.csv(tb2, file = "./1-DE_analysis/snRNAseq-CMPvsGel_DE_gene_list_full.csv", row.names = TRUE)
# filter FDR < 5%, abs(logFC) > log2(1.5) & sort by adj. p-value # keep 1.5 or not
tb2_sig <- filter(tb2, p_adj.loc<=0.05 & abs(logFC) > log2(1.5))
head (tb2_sig)
length(unique(tb2_sig$gene))
| gene | cluster_id | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> | |
| 1 | LOC-Os01g01070 | Meristem | 0.07060 | 0.08700 | 0.000518 | 0.00410 | 0.07690 | 0.00191 | 1.520 | 3.620 | 8.10 | 4.44e-03 | 4.72e-02 | 7.00e-02 | gel-soil |
| 2 | LOC-Os01g01150 | Meristem | 0.13700 | 0.17400 | 0.003630 | 0.00492 | 0.15100 | 0.00413 | 1.470 | 4.630 | 16.10 | 6.13e-05 | 1.49e-03 | 2.51e-03 | gel-soil |
| 3 | LOC-Os01g01450 | Meristem | 0.11400 | 0.12500 | 0.004150 | 0.00246 | 0.11800 | 0.00349 | 1.320 | 4.270 | 10.60 | 1.13e-03 | 1.65e-02 | 2.58e-02 | gel-soil |
| 4 | LOC-Os01g01510 | Meristem | 0.15600 | 0.20200 | 0.008290 | 0.00574 | 0.17400 | 0.00731 | 0.849 | 4.920 | 8.36 | 3.83e-03 | 4.22e-02 | 6.31e-02 | gel-soil |
| 5 | LOC-Os01g01570 | Meristem | 0.15100 | 0.22300 | 0.003630 | 0.00984 | 0.17900 | 0.00604 | 1.240 | 4.940 | 15.70 | 7.58e-05 | 1.79e-03 | 3.00e-03 | gel-soil |
| 6 | LOC-Os01g01870 | Meristem | 0.00229 | 0.00424 | 0.003630 | 0.00574 | 0.00304 | 0.00445 | -4.460 | -0.215 | 47.90 | 4.47e-12 | 4.54e-10 | 9.37e-10 | gel-soil |
# filter out lowly detected gene
tb2_fil <- filter(tb2_sig, gel.frq >=0.1 | soil.frq >=0.1)
head (tb2_fil)
length(unique(tb2_fil$gene))
| gene | cluster_id | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> | |
| 1 | LOC-Os01g01150 | Meristem | 0.1370 | 0.174 | 0.00363 | 0.00492 | 0.151 | 0.00413 | 1.470 | 4.63 | 16.10 | 6.13e-05 | 0.00149 | 0.00251 | gel-soil |
| 2 | LOC-Os01g01450 | Meristem | 0.1140 | 0.125 | 0.00415 | 0.00246 | 0.118 | 0.00349 | 1.320 | 4.27 | 10.60 | 1.13e-03 | 0.01650 | 0.02580 | gel-soil |
| 3 | LOC-Os01g01510 | Meristem | 0.1560 | 0.202 | 0.00829 | 0.00574 | 0.174 | 0.00731 | 0.849 | 4.92 | 8.36 | 3.83e-03 | 0.04220 | 0.06310 | gel-soil |
| 4 | LOC-Os01g01570 | Meristem | 0.1510 | 0.223 | 0.00363 | 0.00984 | 0.179 | 0.00604 | 1.240 | 4.94 | 15.70 | 7.58e-05 | 0.00179 | 0.00300 | gel-soil |
| 5 | LOC-Os01g02890 | Meristem | 0.0952 | 0.120 | 0.00259 | 0.00246 | 0.105 | 0.00254 | 1.590 | 4.08 | 12.00 | 5.36e-04 | 0.00910 | 0.01440 | gel-soil |
| 6 | LOC-Os01g06010 | Meristem | 0.1140 | 0.135 | 0.00467 | 0.00328 | 0.122 | 0.00413 | 1.030 | 4.44 | 8.16 | 4.28e-03 | 0.04590 | 0.06830 | gel-soil |
# label up vs down
tb2_fil <- tb2_fil %>% mutate(up_dn_label = case_when(logFC >=log2(1.5) ~ "Down",
logFC <=log2(1/1.5) ~ "Up",
TRUE ~ "Not DE"))
tb2_fil$clust_up_dn <- paste(tb2_fil$cluster_id, tb2_fil$up_dn_label, sep="_")
head(tb2_fil)
| gene | cluster_id | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast | up_dn_label | clust_up_dn | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> | <chr> | <chr> | |
| 1 | LOC-Os01g01150 | Meristem | 0.1370 | 0.174 | 0.00363 | 0.00492 | 0.151 | 0.00413 | 1.470 | 4.63 | 16.10 | 6.13e-05 | 0.00149 | 0.00251 | gel-soil | Down | Meristem_Down |
| 2 | LOC-Os01g01450 | Meristem | 0.1140 | 0.125 | 0.00415 | 0.00246 | 0.118 | 0.00349 | 1.320 | 4.27 | 10.60 | 1.13e-03 | 0.01650 | 0.02580 | gel-soil | Down | Meristem_Down |
| 3 | LOC-Os01g01510 | Meristem | 0.1560 | 0.202 | 0.00829 | 0.00574 | 0.174 | 0.00731 | 0.849 | 4.92 | 8.36 | 3.83e-03 | 0.04220 | 0.06310 | gel-soil | Down | Meristem_Down |
| 4 | LOC-Os01g01570 | Meristem | 0.1510 | 0.223 | 0.00363 | 0.00984 | 0.179 | 0.00604 | 1.240 | 4.94 | 15.70 | 7.58e-05 | 0.00179 | 0.00300 | gel-soil | Down | Meristem_Down |
| 5 | LOC-Os01g02890 | Meristem | 0.0952 | 0.120 | 0.00259 | 0.00246 | 0.105 | 0.00254 | 1.590 | 4.08 | 12.00 | 5.36e-04 | 0.00910 | 0.01440 | gel-soil | Down | Meristem_Down |
| 6 | LOC-Os01g06010 | Meristem | 0.1140 | 0.135 | 0.00467 | 0.00328 | 0.122 | 0.00413 | 1.030 | 4.44 | 8.16 | 4.28e-03 | 0.04590 | 0.06830 | gel-soil | Down | Meristem_Down |
# Save all the filtered DE genes
write.csv(tb2_fil, file = "./1-DE_analysis/snRNAseq-CMPvsGel_DE_gene_list_q0.05_FC1.5_10percent.csv", row.names = TRUE)
# Number of DEGs per cell type clusters
nb_genes_per_clust <- tb2_fil %>% group_by(cluster_id) %>% tally()
nb_de_gene_count <- tb2_fil %>% group_by(gene) %>% tally(name = "n_clusters_de")
nb_cluster_DE_sig <- left_join(tb2_fil, nb_de_gene_count)
# color up and down
nb_up_dn <- tb2_fil %>% group_by(cluster_id, up_dn_label) %>% tally(name = "n_up_dn") %>% left_join(nb_genes_per_clust)
Joining with `by = join_by(gene)` Joining with `by = join_by(cluster_id)`
# Plot the number of DEGs per cell type clusters
options(repr.plot.width=16, repr.plot.height=16)
nb_v <- ggplot(data=nb_up_dn, aes(x=reorder(cluster_id, n), y=n_up_dn, fill=factor(up_dn_label, levels=c("Up", "Down")))) +
geom_bar(stat="identity", position="stack") + ylab("Number of DEGs") + xlab("") +
scale_y_continuous(limits = c(0, 1500)) +
geom_text(aes(label=n, y = n), hjust=-0.1, vjust=0.5, size=4) +
theme_cowplot() + theme(plot.margin = margin(1,1,1,1, "cm"),
legend.title=element_blank(),
legend.position = c(0.7, 0.5))
(de_up_dn_bar_v <- nb_v + coord_flip())
# Copy tb2_fil for backup
tb3_fil <- tb2_fil
# numbers of Soil induced genes to add as metadata
(Soil_total_n <- tb3_fil %>% select(cluster_id, gene) %>% group_by(cluster_id) %>% tally(name = "total_DE"))
| cluster_id | total_DE |
|---|---|
| <chr> | <int> |
| Atrichoblast | 660 |
| Cortex | 392 |
| Endodermis | 225 |
| Exodermis | 922 |
| Mature root cells | 481 |
| Meristem | 974 |
| Pericycle | 1117 |
| Phloem | 364 |
| Sclerenchyma | 517 |
| Trichoblast | 893 |
| Xylem | 645 |
# numbers of compaction up-regulated genes to add as metadata
(Soil_up_n <- tb3_fil %>% filter(up_dn_label=="Up") %>%
select(cluster_id, gene) %>% group_by(cluster_id) %>% tally(name = "Soil_Up"))
| cluster_id | Soil_Up |
|---|---|
| <chr> | <int> |
| Atrichoblast | 123 |
| Cortex | 38 |
| Endodermis | 14 |
| Exodermis | 123 |
| Mature root cells | 53 |
| Meristem | 261 |
| Pericycle | 225 |
| Phloem | 49 |
| Sclerenchyma | 53 |
| Trichoblast | 80 |
| Xylem | 148 |
# numbers of compaction down-regulated genes to add as metadata
(Soil_dn_n <- tb3_fil %>% filter(up_dn_label=="Down") %>%
select(cluster_id, gene) %>% group_by(cluster_id) %>% tally(name = "Soil_Down"))
| cluster_id | Soil_Down |
|---|---|
| <chr> | <int> |
| Atrichoblast | 537 |
| Cortex | 354 |
| Endodermis | 211 |
| Exodermis | 799 |
| Mature root cells | 428 |
| Meristem | 713 |
| Pericycle | 892 |
| Phloem | 315 |
| Sclerenchyma | 464 |
| Trichoblast | 813 |
| Xylem | 497 |
## all genes background
# DEG results with gene freqs
(res_to_write_frq <- resDS(sce, res, bind = "row", cpm=TRUE, frq=T))
# Output for all background genes with frequency filtering
all_bg <- filter(res_to_write_frq,
gel.frq >=0.1 | soil.frq >=0.1)
| gene | cluster_id | sc_297.cpm | sc_298.cpm | sc_301.cpm | sc_302.cpm | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> |
| LOC-Os01g01010 | Meristem | 11.3000 | 11.1000 | 4.2 | 4.72 | 0.059900 | 0.067000 | 0.001560 | 0.00246 | 0.062600 | 0.001910 | 1.3300 | 3.440 | 5.89000 | 1.52e-02 | 0.11400 | 0.15800 | gel-soil |
| LOC-Os01g01019 | Meristem | 0.3070 | 0.2320 | 1.4 | 0.00 | 0.001910 | 0.001820 | 0.000518 | 0.00000 | 0.001870 | 0.000318 | -1.4100 | -1.260 | 0.52200 | 4.70e-01 | 0.86600 | 0.84900 | gel-soil |
| LOC-Os01g01030 | Meristem | 5.1500 | 5.4600 | 2.8 | 1.57 | 0.030000 | 0.037300 | 0.001040 | 0.00082 | 0.032800 | 0.000953 | 1.2500 | 2.400 | 2.69000 | 1.01e-01 | 0.38300 | 0.45400 | gel-soil |
| LOC-Os01g01040 | Meristem | 21.2000 | 18.7000 | 11.2 | 18.90 | 0.116000 | 0.124000 | 0.004150 | 0.00984 | 0.119000 | 0.006350 | 0.4320 | 4.290 | 1.61000 | 2.04e-01 | 0.57500 | 0.62800 | gel-soil |
| LOC-Os01g01050 | Meristem | 5.6100 | 4.5700 | 2.8 | 3.15 | 0.031900 | 0.031800 | 0.001040 | 0.00164 | 0.031900 | 0.001270 | 0.7880 | 2.370 | 1.26000 | 2.61e-01 | 0.65500 | 0.69400 | gel-soil |
| LOC-Os01g01060 | Meristem | 50.0000 | 57.4000 | 44.8 | 58.20 | 0.223000 | 0.283000 | 0.016100 | 0.02630 | 0.246000 | 0.020000 | 0.0679 | 5.740 | 0.12200 | 7.27e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01070 | Meristem | 12.6000 | 12.9000 | 1.4 | 7.87 | 0.070600 | 0.087000 | 0.000518 | 0.00410 | 0.076900 | 0.001910 | 1.5200 | 3.620 | 8.10000 | 4.44e-03 | 0.04720 | 0.07000 | gel-soil |
| LOC-Os01g01080 | Meristem | 10.0000 | 9.0200 | 7.0 | 4.72 | 0.056800 | 0.064000 | 0.002590 | 0.00246 | 0.059600 | 0.002540 | 0.6850 | 3.240 | 1.77000 | 1.83e-01 | 0.54200 | 0.59200 | gel-soil |
| LOC-Os01g01110 | Meristem | 0.0307 | 0.0000 | 0.0 | 0.00 | 0.000191 | 0.000000 | 0.000000 | 0.00000 | 0.000117 | 0.000000 | 1.6000 | -2.730 | 0.01640 | 8.98e-01 | 0.93000 | 0.94000 | gel-soil |
| LOC-Os01g01115 | Meristem | 0.7670 | 0.6580 | 0.0 | 0.00 | 0.004580 | 0.004850 | 0.000000 | 0.00000 | 0.004680 | 0.000000 | 6.4400 | -0.265 | 1.82000 | 1.77e-01 | 0.53200 | 0.58900 | gel-soil |
| LOC-Os01g01120 | Meristem | 1.1000 | 1.5900 | 0.0 | 0.00 | 0.006300 | 0.011200 | 0.000000 | 0.00000 | 0.008200 | 0.000000 | 7.3200 | 0.505 | 3.51000 | 6.09e-02 | 0.28400 | 0.35100 | gel-soil |
| LOC-Os01g01150 | Meristem | 26.2000 | 27.1000 | 9.8 | 9.44 | 0.137000 | 0.174000 | 0.003630 | 0.00492 | 0.151000 | 0.004130 | 1.4700 | 4.630 | 16.10000 | 6.13e-05 | 0.00149 | 0.00251 | gel-soil |
| LOC-Os01g01160 | Meristem | 1.0700 | 0.9290 | 0.0 | 0.00 | 0.006490 | 0.007280 | 0.000000 | 0.00000 | 0.006790 | 0.000000 | 6.9200 | 0.155 | 2.64000 | 1.04e-01 | 0.38900 | 0.45900 | gel-soil |
| LOC-Os01g01170 | Meristem | 3.5600 | 3.6800 | 0.0 | 1.57 | 0.019500 | 0.025200 | 0.000000 | 0.00082 | 0.021700 | 0.000318 | 2.2700 | 1.860 | 4.15000 | 4.16e-02 | 0.22300 | 0.28500 | gel-soil |
| LOC-Os01g01250 | Meristem | 0.0307 | 0.1550 | 0.0 | 0.00 | 0.000191 | 0.001210 | 0.000000 | 0.00000 | 0.000585 | 0.000000 | 3.5100 | -2.200 | 0.11200 | 7.38e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01280 | Meristem | 0.3990 | 0.4260 | 0.0 | 1.57 | 0.002480 | 0.003340 | 0.000000 | 0.00082 | 0.002810 | 0.000318 | -0.8390 | -0.855 | 0.23500 | 6.28e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01295 | Meristem | 6.7200 | 7.8200 | 4.2 | 4.72 | 0.037400 | 0.054000 | 0.001560 | 0.00246 | 0.043800 | 0.001910 | 0.7010 | 2.850 | 1.43000 | 2.32e-01 | 0.61600 | 0.65700 | gel-soil |
| LOC-Os01g01302 | Meristem | 2.8800 | 3.0200 | 0.0 | 1.57 | 0.015100 | 0.021500 | 0.000000 | 0.00082 | 0.017600 | 0.000318 | 1.9800 | 1.580 | 2.94000 | 8.63e-02 | 0.35300 | 0.42000 | gel-soil |
| LOC-Os01g01307 | Meristem | 25.1000 | 27.3000 | 26.6 | 25.20 | 0.129000 | 0.161000 | 0.009850 | 0.01230 | 0.142000 | 0.010800 | 0.0111 | 4.710 | 0.00168 | 9.67e-01 | 0.97800 | 0.98000 | gel-soil |
| LOC-Os01g01312 | Meristem | 6.0100 | 5.6500 | 7.0 | 3.15 | 0.033600 | 0.038500 | 0.002590 | 0.00164 | 0.035500 | 0.002220 | 0.1720 | 2.570 | 0.08850 | 7.66e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01360 | Meristem | 0.1840 | 0.0774 | 0.0 | 0.00 | 0.000954 | 0.000606 | 0.000000 | 0.00000 | 0.000820 | 0.000000 | 4.1000 | -1.900 | 0.20300 | 6.52e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01380 | Meristem | 1.9600 | 2.6300 | 0.0 | 0.00 | 0.011300 | 0.019400 | 0.000000 | 0.00000 | 0.014400 | 0.000000 | 8.0900 | 1.220 | 5.86000 | 1.55e-02 | 0.11600 | 0.15900 | gel-soil |
| LOC-Os01g01390 | Meristem | 6.1600 | 6.4200 | 5.6 | 4.72 | 0.035900 | 0.046100 | 0.002070 | 0.00164 | 0.039800 | 0.001910 | 0.2770 | 2.670 | 0.23500 | 6.28e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01400 | Meristem | 1.6300 | 2.0500 | 2.8 | 0.00 | 0.009350 | 0.014900 | 0.001040 | 0.00000 | 0.011500 | 0.000635 | 0.2980 | 0.959 | 0.08780 | 7.67e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01420 | Meristem | 3.3400 | 3.7500 | 2.8 | 3.15 | 0.019600 | 0.026100 | 0.001040 | 0.00164 | 0.022100 | 0.001270 | 0.2530 | 1.870 | 0.11700 | 7.32e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01430 | Meristem | 0.0307 | 0.0387 | 0.0 | 0.00 | 0.000191 | 0.000303 | 0.000000 | 0.00000 | 0.000234 | 0.000000 | 2.3500 | -2.580 | 0.03590 | 8.50e-01 | 0.93000 | 0.91500 | gel-soil |
| LOC-Os01g01450 | Meristem | 21.4000 | 19.3000 | 11.2 | 4.72 | 0.114000 | 0.125000 | 0.004150 | 0.00246 | 0.118000 | 0.003490 | 1.3200 | 4.270 | 10.60000 | 1.13e-03 | 0.01650 | 0.02580 | gel-soil |
| LOC-Os01g01510 | Meristem | 30.9000 | 33.2000 | 23.8 | 11.00 | 0.156000 | 0.202000 | 0.008290 | 0.00574 | 0.174000 | 0.007310 | 0.8490 | 4.920 | 8.36000 | 3.83e-03 | 0.04220 | 0.06310 | gel-soil |
| LOC-Os01g01570 | Meristem | 30.4000 | 36.2000 | 9.8 | 18.90 | 0.151000 | 0.223000 | 0.003630 | 0.00984 | 0.179000 | 0.006040 | 1.2400 | 4.940 | 15.70000 | 7.58e-05 | 0.00179 | 0.00300 | gel-soil |
| LOC-Os01g01580 | Meristem | 1.2300 | 1.2800 | 0.0 | 0.00 | 0.007440 | 0.009100 | 0.000000 | 0.00000 | 0.008080 | 0.000000 | 7.2300 | 0.430 | 3.30000 | 6.91e-02 | 0.30800 | 0.37500 | gel-soil |
| ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ | ⋮ |
| LOC-Os07g27290 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os07g44110 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os07g06920 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os07g14514 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os07g25330 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os07g29900 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os08g18810 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os08g35820 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os08g36920 | Mature root cells | 0 | 0.00 | 24.10 | 17.40 | 0 | 0.000 | 0.00577 | 0.00831 | 0.0000 | 0.006640 | -5.71 | 3.88 | 13.200 | 0.000282 | 0.0131 | 0.00868 | gel-soil |
| LOC-Os08g07140 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os08g25860 | Mature root cells | 0 | 2.24 | 0.00 | 0.00 | 0 | 0.027 | 0.00000 | 0.00000 | 0.0133 | 0.000000 | 2.02 | 2.89 | 0.205 | 0.651000 | 0.8160 | 0.91500 | gel-soil |
| LOC-Os10g26010 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os11g02880 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os03g05930 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os06g11550 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os06g20750 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os10g32100 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os02g16830 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os03g36239 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os01g50670 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os10g05740 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os10g37560 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os04g49810 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os05g24520 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os05g49090 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os09g03830 | Mature root cells | 0 | 0.00 | 4.02 | 0.00 | 0 | 0.000 | 0.00144 | 0.00000 | 0.0000 | 0.000949 | -2.74 | 2.91 | 0.517 | 0.472000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os03g25010 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os04g06400 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os06g21440 | Mature root cells | 0 | 0.00 | 0.00 | 5.79 | 0 | 0.000 | 0.00000 | 0.00277 | 0.0000 | 0.000949 | -2.77 | 2.91 | 0.532 | 0.466000 | 0.8160 | 0.84900 | gel-soil |
| LOC-Os09g39170 | Mature root cells | 0 | 0.00 | 0.00 | 17.40 | 0 | 0.000 | 0.00000 | 0.00831 | 0.0000 | 0.002850 | -4.23 | 3.22 | 3.420 | 0.064400 | 0.5080 | 0.36200 | gel-soil |
# Get the total gene detected for each cluster
(bg_n <- all_bg %>%
select(cluster_id, gene) %>%
group_by(cluster_id) %>%
tally(name = "All_genes"))
| cluster_id | All_genes |
|---|---|
| <chr> | <int> |
| Atrichoblast | 1654 |
| Cortex | 1853 |
| Endodermis | 2777 |
| Exodermis | 2181 |
| Mature root cells | 3804 |
| Meristem | 2180 |
| Pericycle | 2120 |
| Phloem | 2313 |
| Sclerenchyma | 1877 |
| Trichoblast | 2298 |
| Xylem | 2056 |
# Make a metadata for UMAP presentation
(de_meta_for_umap <- data.frame("barcode"=colnames(rc.comparison),
"cluster_id"=rc.comparison$manual.anno))
| barcode | cluster_id | |
|---|---|---|
| <chr> | <fct> | |
| AAACCCAAGATGAATC_1 | AAACCCAAGATGAATC_1 | Exodermis |
| AAACCCAAGGACGCTA_1 | AAACCCAAGGACGCTA_1 | Pericycle |
| AAACCCAAGGATATGT_1 | AAACCCAAGGATATGT_1 | Atrichoblast |
| AAACCCAAGGCACCAA_1 | AAACCCAAGGCACCAA_1 | Cortex |
| AAACCCAAGTCCCTAA_1 | AAACCCAAGTCCCTAA_1 | Exodermis |
| AAACCCACAACCCGCA_1 | AAACCCACAACCCGCA_1 | Meristem |
| AAACCCACAACTCCCT_1 | AAACCCACAACTCCCT_1 | Exodermis |
| AAACCCACAATTGCTG_1 | AAACCCACAATTGCTG_1 | Sclerenchyma |
| AAACCCACACATATCG_1 | AAACCCACACATATCG_1 | Atrichoblast |
| AAACCCACAGACAAAT_1 | AAACCCACAGACAAAT_1 | Meristem |
| AAACCCACAGCAAGAC_1 | AAACCCACAGCAAGAC_1 | Meristem |
| AAACCCACAGTAGATA_1 | AAACCCACAGTAGATA_1 | Meristem |
| AAACCCACATCGTGGC_1 | AAACCCACATCGTGGC_1 | Meristem |
| AAACCCAGTGAGATAT_1 | AAACCCAGTGAGATAT_1 | Meristem |
| AAACCCAGTGTAACGG_1 | AAACCCAGTGTAACGG_1 | Sclerenchyma |
| AAACCCAGTTCCAGGC_1 | AAACCCAGTTCCAGGC_1 | Pericycle |
| AAACCCATCTGAGAAA_1 | AAACCCATCTGAGAAA_1 | Pericycle |
| AAACGAAAGAGTCAGC_1 | AAACGAAAGAGTCAGC_1 | Trichoblast |
| AAACGAAAGAGTCCGA_1 | AAACGAAAGAGTCCGA_1 | Trichoblast |
| AAACGAAAGCTAGATA_1 | AAACGAAAGCTAGATA_1 | Meristem |
| AAACGAAAGGAACGCT_1 | AAACGAAAGGAACGCT_1 | Meristem |
| AAACGAACACGAGAAC_1 | AAACGAACACGAGAAC_1 | Meristem |
| AAACGAAGTGGTTTAC_1 | AAACGAAGTGGTTTAC_1 | Meristem |
| AAACGAATCACGGAGA_1 | AAACGAATCACGGAGA_1 | Pericycle |
| AAACGAATCATGAGTC_1 | AAACGAATCATGAGTC_1 | Meristem |
| AAACGAATCCCAGTGG_1 | AAACGAATCCCAGTGG_1 | Meristem |
| AAACGCTCAAAGCACG_1 | AAACGCTCAAAGCACG_1 | Meristem |
| AAACGCTCAACTAGAA_1 | AAACGCTCAACTAGAA_1 | Meristem |
| AAACGCTCACAGTCAT_1 | AAACGCTCACAGTCAT_1 | Exodermis |
| AAACGCTCACATTCGA_1 | AAACGCTCACATTCGA_1 | Meristem |
| ⋮ | ⋮ | ⋮ |
| TTTGACTGTATGTCTG_8 | TTTGACTGTATGTCTG_8 | Xylem |
| TTTGACTGTCCAAATC_8 | TTTGACTGTCCAAATC_8 | Pericycle |
| TTTGACTGTGTCTCCT_8 | TTTGACTGTGTCTCCT_8 | Exodermis |
| TTTGACTTCAGACATC_8 | TTTGACTTCAGACATC_8 | Pericycle |
| TTTGACTTCCATCTAT_8 | TTTGACTTCCATCTAT_8 | Sclerenchyma |
| TTTGACTTCCCGAGAC_8 | TTTGACTTCCCGAGAC_8 | Atrichoblast |
| TTTGACTTCCGATTAG_8 | TTTGACTTCCGATTAG_8 | Exodermis |
| TTTGGAGCACTATCCC_8 | TTTGGAGCACTATCCC_8 | Pericycle |
| TTTGGTTAGCCACCGT_8 | TTTGGTTAGCCACCGT_8 | Sclerenchyma |
| TTTGGTTAGGCCTTGC_8 | TTTGGTTAGGCCTTGC_8 | Pericycle |
| TTTGGTTAGTAACCGG_8 | TTTGGTTAGTAACCGG_8 | Atrichoblast |
| TTTGGTTCAACACACT_8 | TTTGGTTCAACACACT_8 | Exodermis |
| TTTGGTTCACACACTA_8 | TTTGGTTCACACACTA_8 | Meristem |
| TTTGGTTCACATTGTG_8 | TTTGGTTCACATTGTG_8 | Meristem |
| TTTGGTTCACTTCCTG_8 | TTTGGTTCACTTCCTG_8 | Trichoblast |
| TTTGGTTCAGCTATAC_8 | TTTGGTTCAGCTATAC_8 | Xylem |
| TTTGGTTCATGACTTG_8 | TTTGGTTCATGACTTG_8 | Sclerenchyma |
| TTTGGTTGTAACCCTA_8 | TTTGGTTGTAACCCTA_8 | Phloem |
| TTTGGTTGTATGAGCG_8 | TTTGGTTGTATGAGCG_8 | Phloem |
| TTTGGTTGTCTAACGT_8 | TTTGGTTGTCTAACGT_8 | Exodermis |
| TTTGGTTTCACTAGCA_8 | TTTGGTTTCACTAGCA_8 | Sclerenchyma |
| TTTGGTTTCAGTCTTT_8 | TTTGGTTTCAGTCTTT_8 | Pericycle |
| TTTGGTTTCATTTGTC_8 | TTTGGTTTCATTTGTC_8 | Xylem |
| TTTGTTGGTCAACCAT_8 | TTTGTTGGTCAACCAT_8 | Pericycle |
| TTTGTTGTCATTACTC_8 | TTTGTTGTCATTACTC_8 | Endodermis |
| TTTGTTGTCCGCTGTT_8 | TTTGTTGTCCGCTGTT_8 | Trichoblast |
| TTTGTTGTCCTACCGT_8 | TTTGTTGTCCTACCGT_8 | Sclerenchyma |
| TTTGTTGTCGGTCATA_8 | TTTGTTGTCGGTCATA_8 | Mature root cells |
| TTTGTTGTCTCGGTCT_8 | TTTGTTGTCTCGGTCT_8 | Phloem |
| TTTGTTGTCTGATTCT_8 | TTTGTTGTCTGATTCT_8 | Phloem |
# Add the quantified DEG number into the metadata
de_meta_for_umap <- left_join(de_meta_for_umap, bg_n)
de_meta_for_umap <- left_join(de_meta_for_umap, Soil_total_n)
de_meta_for_umap <- left_join(de_meta_for_umap, Soil_up_n)
de_meta_for_umap <- left_join(de_meta_for_umap, Soil_dn_n)
head(de_meta_for_umap, n =7)
Joining with `by = join_by(cluster_id)` Joining with `by = join_by(cluster_id)` Joining with `by = join_by(cluster_id)` Joining with `by = join_by(cluster_id)`
| barcode | cluster_id | All_genes | total_DE | Soil_Up | Soil_Down | |
|---|---|---|---|---|---|---|
| <chr> | <chr> | <int> | <int> | <int> | <int> | |
| 1 | AAACCCAAGATGAATC_1 | Exodermis | 2181 | 922 | 123 | 799 |
| 2 | AAACCCAAGGACGCTA_1 | Pericycle | 2120 | 1117 | 225 | 892 |
| 3 | AAACCCAAGGATATGT_1 | Atrichoblast | 1654 | 660 | 123 | 537 |
| 4 | AAACCCAAGGCACCAA_1 | Cortex | 1853 | 392 | 38 | 354 |
| 5 | AAACCCAAGTCCCTAA_1 | Exodermis | 2181 | 922 | 123 | 799 |
| 6 | AAACCCACAACCCGCA_1 | Meristem | 2180 | 974 | 261 | 713 |
| 7 | AAACCCACAACTCCCT_1 | Exodermis | 2181 | 922 | 123 | 799 |
# replace NA with zero for catagories that were too small to test
de_meta_for_umap[is.na(de_meta_for_umap)] <- 0
# Calculate the percentage for DEG number
de_meta_for_umap <- mutate(de_meta_for_umap,
Percent_DE=(total_DE/All_genes)*100,
Percent_Up=(Soil_Up/All_genes)*100,
Percent_Dn=(Soil_Down/All_genes)*100)
head(de_meta_for_umap, n =10)
| barcode | cluster_id | All_genes | total_DE | Soil_Up | Soil_Down | Percent_DE | Percent_Up | Percent_Dn | |
|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <int> | <int> | <int> | <int> | <dbl> | <dbl> | <dbl> | |
| 1 | AAACCCAAGATGAATC_1 | Exodermis | 2181 | 922 | 123 | 799 | 42.27419 | 5.639615 | 36.63457 |
| 2 | AAACCCAAGGACGCTA_1 | Pericycle | 2120 | 1117 | 225 | 892 | 52.68868 | 10.613208 | 42.07547 |
| 3 | AAACCCAAGGATATGT_1 | Atrichoblast | 1654 | 660 | 123 | 537 | 39.90326 | 7.436518 | 32.46675 |
| 4 | AAACCCAAGGCACCAA_1 | Cortex | 1853 | 392 | 38 | 354 | 21.15488 | 2.050729 | 19.10416 |
| 5 | AAACCCAAGTCCCTAA_1 | Exodermis | 2181 | 922 | 123 | 799 | 42.27419 | 5.639615 | 36.63457 |
| 6 | AAACCCACAACCCGCA_1 | Meristem | 2180 | 974 | 261 | 713 | 44.67890 | 11.972477 | 32.70642 |
| 7 | AAACCCACAACTCCCT_1 | Exodermis | 2181 | 922 | 123 | 799 | 42.27419 | 5.639615 | 36.63457 |
| 8 | AAACCCACAATTGCTG_1 | Sclerenchyma | 1877 | 517 | 53 | 464 | 27.54395 | 2.823655 | 24.72030 |
| 9 | AAACCCACACATATCG_1 | Atrichoblast | 1654 | 660 | 123 | 537 | 39.90326 | 7.436518 | 32.46675 |
| 10 | AAACCCACAGACAAAT_1 | Meristem | 2180 | 974 | 261 | 713 | 44.67890 | 11.972477 | 32.70642 |
total_de_meta <- de_meta_for_umap$total_DE
names(total_de_meta) <- colnames(rc.comparison)
rc.comparison <- AddMetaData(
object = rc.comparison,
metadata = total_de_meta,
col.name = 'total_DE')
up_de_meta <- de_meta_for_umap$Soil_Up
names(up_de_meta) <- colnames(rc.comparison)
rc.comparison <- AddMetaData(
object = rc.comparison,
metadata = up_de_meta,
col.name = 'Soil_Up')
dn_de_meta <- de_meta_for_umap$Soil_Down
names(dn_de_meta) <- colnames(rc.comparison)
rc.comparison <- AddMetaData(
object = rc.comparison,
metadata = dn_de_meta,
col.name = 'Soil_Down')
options(repr.plot.width=16, repr.plot.height=16)
FeaturePlot(rc.comparison,
features = c("total_DE"),
max.cutoff = 1000,
min.cutoff = 600,
label=F,
order = T,
cols=c("beige", "#01665e"),
pt.size = 0.5) +
ggtitle("Total Com DE genes") +
theme_void(base_size = 16) +
theme(plot.title = element_text(hjust = 0.5)) +
labs(color="# genes") + theme(plot.margin = margin(0.5,0.5,0.5,0.5, "cm"))
## GO term analysis for DEGs
#
markers_sel_all <- select(tb2_fil, gene, cluster_id)
markers_list_all <- split(markers_sel_all, f=markers_sel_all$cluster_id)
#this makes list from long df of gene lists - TARGET is what we want to keep
markers_list_all <- lapply(markers_list_all, function(x) x[names(x)=="gene"])
# convert each sublist into character and eliminate duplicates
markers_list_all <- lapply(markers_list_all, function(x) as.character(unique(x$gene)))
# Change the format of gene ID to make it recognizable: change"-" to "_"
for (i in 1:length(markers_list_all)){
markers_list_all[[i]] <- gsub("LOC-","LOC_",markers_list_all[[i]])
}
head(markers_list_all, n=1)
# Remove all the gene IDs which does not follow "LOC_OS" format
for (i in 1:length(markers_list_all)){
markers_list_all[[i]] <- markers_list_all[[i]][grep("LOC_",markers_list_all[[i]])]
}
# Load the MSU-RAP ID converter table downloaded from https://rapdb.dna.affrc.go.jp/download/irgsp1.html
msu2rap <- read.table("./ID_conversion/RAP-MSU_2023-03-15.txt.gz")
# Convert the MSU ID to RAP ID if the MSU ID can be found in V2 column in msu2rap table
# grep does not require exact mtach
markers_list_all2 <- markers_list_all
for (i in 1:length(markers_list_all2)){
for (j in 1:length(markers_list_all2[[i]])){
suppressMessages(markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j],msu2rap$V2)])
}
}
## There will be warning message because RAP and MSU are not strictly one to one
Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list_all2[[i]][j] <- msu2rap$V1[grep(markers_list_all2[[i]][j], : “number of items to replace is not a multiple of replacement length”
#Check whether conversion works
markers_list_all2[[1]]
# filter out the "None" value
markers_list_all3 <- markers_list_all2
for (i in 1:length(markers_list_all3)){
markers_list_all3[[i]] <- markers_list_all3[[i]][grep("Os",markers_list_all3[[i]])]
}
markers_list_all3[[1]]
# Run GO term analysis with gprofiler2 gost function
cluster_GO_all <- gost(markers_list_all3, organism = "osativa", correction_method = "fdr", significant = FALSE, multi_query = FALSE)
cluster_GO_all_df <- cluster_GO_all[[1]]
cluster_GO_all_sig <- filter(cluster_GO_all_df, p_value<=0.01)
head(cluster_GO_all_sig, n=5)
| query | significant | p_value | term_size | query_size | intersection_size | precision | recall | term_id | source | term_name | effective_domain_size | source_order | parents | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <lgl> | <dbl> | <int> | <int> | <int> | <dbl> | <dbl> | <chr> | <chr> | <chr> | <int> | <int> | <list> | |
| 1 | Atrichoblast | TRUE | 0.002319737 | 120 | 358 | 13 | 0.03631285 | 0.10833333 | GO:0009205 | GO:BP | purine ribonucleoside triphosphate metabolic process | 15948 | 3408 | GO:00091.... |
| 2 | Atrichoblast | TRUE | 0.002319737 | 121 | 358 | 13 | 0.03631285 | 0.10743802 | GO:0009144 | GO:BP | purine nucleoside triphosphate metabolic process | 15948 | 3348 | GO:0009141 |
| 3 | Atrichoblast | TRUE | 0.003422468 | 132 | 358 | 13 | 0.03631285 | 0.09848485 | GO:0009199 | GO:BP | ribonucleoside triphosphate metabolic process | 15948 | 3402 | GO:0009141 |
| 4 | Atrichoblast | TRUE | 0.003422468 | 114 | 358 | 12 | 0.03351955 | 0.10526316 | GO:0046034 | GO:BP | ATP metabolic process | 15948 | 12209 | GO:00091.... |
| 5 | Atrichoblast | TRUE | 0.004201293 | 140 | 358 | 13 | 0.03631285 | 0.09285714 | GO:0009141 | GO:BP | nucleoside triphosphate metabolic process | 15948 | 3345 | GO:0006753 |
# Save the GO term analysis result
cluster_GO_all_save <- as.matrix (cluster_GO_all_sig)
write.csv(cluster_GO_all_save, file = "./1-DE_analysis/snRNA-seq_CMPvsGel_DE-combined_gene_GO_analysis.csv", row.names = TRUE)
# Save the GO term analysis result
cluster_GO_all_df <- as.matrix (cluster_GO_all_df)
write.csv(cluster_GO_all_df, file = "./1-DE_analysis/snRNA-seq_CMPvsGel_DE-combined_gene_GO_analysis-full.csv", row.names = TRUE)
## GO term analysis for DEGs
#
markers_sel <- select(tb2_fil, gene, clust_up_dn)
markers_list <- split(markers_sel, f=markers_sel$clust_up_dn)
#this makes list from long df of gene lists - TARGET is what we want to keep
markers_list <- lapply(markers_list, function(x) x[names(x)=="gene"])
# convert each sublist into character and eliminate duplicates
markers_list <- lapply(markers_list, function(x) as.character(unique(x$gene)))
head(tb2_fil)
| gene | cluster_id | sc_297.frq | sc_298.frq | sc_301.frq | sc_302.frq | gel.frq | soil.frq | logFC | logCPM | F | p_val | p_adj.loc | p_adj.glb | contrast | up_dn_label | clust_up_dn | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <chr> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <dbl> | <chr> | <chr> | <chr> | |
| 1 | LOC-Os01g01150 | Meristem | 0.1370 | 0.174 | 0.00363 | 0.00492 | 0.151 | 0.00413 | 1.470 | 4.63 | 16.10 | 6.13e-05 | 0.00149 | 0.00251 | gel-soil | Down | Meristem_Down |
| 2 | LOC-Os01g01450 | Meristem | 0.1140 | 0.125 | 0.00415 | 0.00246 | 0.118 | 0.00349 | 1.320 | 4.27 | 10.60 | 1.13e-03 | 0.01650 | 0.02580 | gel-soil | Down | Meristem_Down |
| 3 | LOC-Os01g01510 | Meristem | 0.1560 | 0.202 | 0.00829 | 0.00574 | 0.174 | 0.00731 | 0.849 | 4.92 | 8.36 | 3.83e-03 | 0.04220 | 0.06310 | gel-soil | Down | Meristem_Down |
| 4 | LOC-Os01g01570 | Meristem | 0.1510 | 0.223 | 0.00363 | 0.00984 | 0.179 | 0.00604 | 1.240 | 4.94 | 15.70 | 7.58e-05 | 0.00179 | 0.00300 | gel-soil | Down | Meristem_Down |
| 5 | LOC-Os01g02890 | Meristem | 0.0952 | 0.120 | 0.00259 | 0.00246 | 0.105 | 0.00254 | 1.590 | 4.08 | 12.00 | 5.36e-04 | 0.00910 | 0.01440 | gel-soil | Down | Meristem_Down |
| 6 | LOC-Os01g06010 | Meristem | 0.1140 | 0.135 | 0.00467 | 0.00328 | 0.122 | 0.00413 | 1.030 | 4.44 | 8.16 | 4.28e-03 | 0.04590 | 0.06830 | gel-soil | Down | Meristem_Down |
length(markers_list)
# Change the format of gene ID to make it recognizable: change"-" to "_"
for (i in 1:length(markers_list)){
markers_list[[i]] <- gsub("LOC-","LOC_",markers_list[[i]])
}
head(markers_list, n=1)
# Remove all the gene IDs which does not follow "LOC_OS" format
for (i in 1:length(markers_list)){
markers_list[[i]] <- markers_list[[i]][grep("LOC_",markers_list[[i]])]
}
# Load the MSU-RAP ID converter table downloaded from https://rapdb.dna.affrc.go.jp/download/irgsp1.html
msu2rap <- read.table("./ID_conversion/RAP-MSU_2023-03-15.txt.gz")
head(msu2rap, n=5)
| V1 | V2 | |
|---|---|---|
| <chr> | <chr> | |
| 1 | Os01g0100100 | LOC_Os01g01010.1,LOC_Os01g01010.2 |
| 2 | Os01g0100200 | LOC_Os01g01019.1 |
| 3 | Os01g0100300 | None |
| 4 | Os01g0100400 | LOC_Os01g01030.1 |
| 5 | Os01g0100466 | None |
# Convert the MSU ID to RAP ID if the MSU ID can be found in V2 column in msu2rap table
# grep does not require exact mtach
markers_list2 <- markers_list
for (i in 1:length(markers_list2)){
for (j in 1:length(markers_list2[[i]])){
suppressMessages(markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j],msu2rap$V2)])
}
}
## There will be warning message because RAP and MSU are not strictly one to one
Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length” Warning message in markers_list2[[i]][j] <- msu2rap$V1[grep(markers_list2[[i]][j], : “number of items to replace is not a multiple of replacement length”
#Check whether conversion works
markers_list2[[14]]
# filter out the "None" value
markers_list3 <- markers_list2
for (i in 1:length(markers_list3)){
markers_list3[[i]] <- markers_list3[[i]][grep("Os",markers_list3[[i]])]
}
markers_list3[[14]]
# Run GO term analysis with gprofiler2 gost function
cluster_GO <- gost(markers_list3, organism = "osativa", correction_method = "fdr", significant = FALSE, multi_query = FALSE)
cluster_GO_df <- cluster_GO[[1]]
cluster_GO_sig <- filter(cluster_GO_df, p_value<=0.01)
head(cluster_GO_sig, n=5)
| query | significant | p_value | term_size | query_size | intersection_size | precision | recall | term_id | source | term_name | effective_domain_size | source_order | parents | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| <chr> | <lgl> | <dbl> | <int> | <int> | <int> | <dbl> | <dbl> | <chr> | <chr> | <chr> | <int> | <int> | <list> | |
| 1 | Atrichoblast_Down | TRUE | 4.609595e-03 | 46 | 85 | 7 | 0.08235294 | 0.15217391 | KEGG:00999 | KEGG | Biosynthesis of various plant secondary metabolites | 3695 | 162 | KEGG:00000 |
| 2 | Atrichoblast_Down | TRUE | 6.360983e-03 | 38 | 85 | 6 | 0.07058824 | 0.15789474 | KEGG:00460 | KEGG | Cyanoamino acid metabolism | 3695 | 59 | KEGG:00000 |
| 3 | Atrichoblast_Up | TRUE | 5.543150e-12 | 121 | 78 | 13 | 0.16666667 | 0.10743802 | GO:0009144 | GO:BP | purine nucleoside triphosphate metabolic process | 15948 | 3348 | GO:0009141 |
| 4 | Atrichoblast_Up | TRUE | 5.543150e-12 | 120 | 78 | 13 | 0.16666667 | 0.10833333 | GO:0009205 | GO:BP | purine ribonucleoside triphosphate metabolic process | 15948 | 3408 | GO:00091.... |
| 5 | Atrichoblast_Up | TRUE | 1.163328e-11 | 132 | 78 | 13 | 0.16666667 | 0.09848485 | GO:0009199 | GO:BP | ribonucleoside triphosphate metabolic process | 15948 | 3402 | GO:0009141 |
## Save the whole GO list
cluster_GO_full_save <- as.matrix (cluster_GO_df)
write.csv(cluster_GO_full_save, file = "./1-DE_analysis/snRNA-seq_CMPvsGel_DE_gene_GO_analysis-full.csv", row.names = TRUE)
# Save the GO term analysis result
cluster_GO_save <- as.matrix (cluster_GO_sig)
write.csv(cluster_GO_save, file = "./1-DE_analysis/snRNA-seq_CMPvsGel_DE_gene_GO_analysis.csv", row.names = TRUE)
# Read the defense relevant gene list
defense.gene <- read.csv("./1-DE_analysis/Biology_process_genes//Defense_genes.csv")
defense.gene$Gene.ID
# Check Defense relevant genes
DefaultAssay(rc.comparison) <- "SCT"
options(repr.plot.width=12, repr.plot.height=6)
FeaturePlot(rc.comparison, features = "LOC-Os11g45160",
cols = c("grey", "red"), label=F, repel=F, pt.size = 0.8,
label.size = 3.5, order=T, split.by="normal")
Warning message in FeaturePlot(rc.comparison, features = "LOC-Os11g45160", cols = c("grey", :
“All cells have the same value (0) of LOC-Os11g45160.”
# Check UMI counts in each seurat cluster
options(repr.plot.width=36, repr.plot.height=36)
VlnPlot(object = rc.comparison, features = "nCount_RNA", group.by = "manual.anno")
table(rc.comparison$normal)
gel soil 16219 13838
## Subgroup the "normal" groups for the following analysis
rc.gel <- subset(rc.comparison, cells=colnames(rc.comparison)[which(rc.comparison$normal!="soil")])
# Check UMI counts in each seurat cluster
options(repr.plot.width=12, repr.plot.height=18)
VlnPlot(object = rc.gel, features = "nCount_RNA", group.by = "manual.anno",
pt.size = 0.1)
## Subgroup the "normal" groups for the following analysis
rc.soil <- subset(rc.comparison, cells=colnames(rc.comparison)[which(rc.comparison$normal!="gel")])
# Check UMI counts in each seurat cluster
options(repr.plot.width=12, repr.plot.height=18)
VlnPlot(object = rc.soil, features = "nCount_RNA", group.by = "manual.anno",
pt.size = 0.1)
table(rc.integrated$gel_soil)
non-compact soil compact soil gel
13838 18390 22797
## Subgroup the "normal" groups for the following analysis
rc.cmp <- subset(rc.integrated, cells=colnames(rc.integrated)[which(rc.integrated$gel_soil == "compact soil")])
# Check UMI counts in each seurat cluster
options(repr.plot.width=12, repr.plot.height=18)
VlnPlot(object = rc.cmp, features = "nCount_RNA", group.by = "manual.anno",
pt.size = 0.1)
# Read the defense relevant gene list
defense.gene <- read.csv("./1-DE_analysis/Biology_process_genes//Defense_genes.csv")
defense.gene$Gene.ID
## Check gene existence in single cell data-exodermis
match(defense.gene$Gene.ID,rownames(rc.exodermis@assays$SCT@data))
# Read the vesicle-mediated transport relevant gene list
transport.gene <- read.csv("./1-DE_analysis/Biology_process_genes//Transport_genes.csv")
transport.gene$Gene.ID
## Check gene existence in single cell data
match(transport.gene$Gene.ID,rownames(rc.exodermis@assays$SCT@data))
# Check DE gene expression-defense
DefaultAssay(rc.comparison) <- "SCT"
options(repr.plot.width=12, repr.plot.height=6)
FeaturePlot(rc.comparison, features = "LOC-Os07g14540",
cols = c("grey", "red"), label=F, repel=F, pt.size = 0.8,
label.size = 3.5, order=T, split.by="condition")
# Check the Trichoblast markers-Correlation analysis
options(repr.plot.width=18, repr.plot.height=42)
DefaultAssay(rc.integrated) <- "SCT"
FeaturePlot(rc.integrated, cols = c("grey", "#Ff4061"),
feature = c("LOC-Os07g01420","LOC-Os01g07060",
"LOC-Os05g38770","LOC-Os06g08500","LOC-Os01g11750",
"LOC-Os01g07030","LOC-Os06g01920","LOC-Os07g30090",
"LOC-Os04g46910","LOC-Os07g26460","LOC-Os12g05380",
"LOC-Os02g18430","LOC-Os07g01420","LOC-Os07g30090",
"LOC-Os05g35810","LOC-Os01g18970","LOC-Os06g02390",
"LOC-Os01g13120","LOC-Os03g04210","LOC-Os02g17880"),
order = T, ncol = 3)
# Check all the packages versions
sessionInfo()
R version 4.2.0 (2022-04-22) Platform: x86_64-conda-linux-gnu (64-bit) Running under: CentOS Stream 8 Matrix products: default BLAS/LAPACK: /hpc/group/pbenfeylab/mz187/miniconda3/envs/muscat/lib/libopenblasp-r0.3.21.so locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=en_US.UTF-8 LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] stats4 grid stats graphics grDevices utils datasets [8] methods base other attached packages: [1] future_1.32.0 scran_1.26.0 [3] scuttle_1.8.0 SingleCellExperiment_1.20.0 [5] SummarizedExperiment_1.28.0 Biobase_2.58.0 [7] GenomicRanges_1.50.0 GenomeInfoDb_1.34.9 [9] IRanges_2.32.0 S4Vectors_0.36.0 [11] BiocGenerics_0.44.0 MatrixGenerics_1.10.0 [13] matrixStats_1.0.0 limma_3.54.0 [15] muscat_1.12.0 ggrepel_0.9.3 [17] gprofiler2_0.2.2 GeneOverlap_1.34.0 [19] circlize_0.4.15 ComplexHeatmap_2.14.0 [21] cowplot_1.1.1 Seurat_3.1.5 [23] lubridate_1.9.2 forcats_1.0.0 [25] stringr_1.5.0 dplyr_1.1.2 [27] purrr_1.0.1 readr_2.1.4 [29] tidyr_1.3.0 tibble_3.2.1 [31] ggplot2_3.4.2 tidyverse_2.0.0 loaded via a namespace (and not attached): [1] utf8_1.2.3 reticulate_1.25 [3] tidyselect_1.2.0 lme4_1.1-33 [5] RSQLite_2.3.1 AnnotationDbi_1.60.0 [7] htmlwidgets_1.6.2 BiocParallel_1.32.5 [9] Rtsne_0.16 ScaledMatrix_1.6.0 [11] munsell_0.5.0 codetools_0.2-19 [13] ica_1.0-3 statmod_1.4.36 [15] pbdZMQ_0.3-9 withr_2.5.0 [17] colorspace_2.1-0 uuid_1.1-0 [19] ROCR_1.0-11 listenv_0.9.0 [21] Rdpack_2.4 repr_1.1.6 [23] GenomeInfoDbData_1.2.9 bit64_4.0.5 [25] glmmTMB_1.1.7 parallelly_1.36.0 [27] vctrs_0.6.3 generics_0.1.3 [29] timechange_0.2.0 R6_2.5.1 [31] doParallel_1.0.17 ggbeeswarm_0.7.2 [33] clue_0.3-64 rsvd_1.0.5 [35] locfit_1.5-9.8 bitops_1.0-7 [37] cachem_1.0.8 DelayedArray_0.24.0 [39] scales_1.2.1 beeswarm_0.4.0 [41] gtable_0.3.3 beachmat_2.14.0 [43] globals_0.16.2 rlang_1.1.1 [45] genefilter_1.80.0 GlobalOptions_0.1.2 [47] splines_4.2.0 TMB_1.9.4 [49] lazyeval_0.2.2 broom_1.0.5 [51] reshape2_1.4.4 backports_1.4.1 [53] tools_4.2.0 gplots_3.1.3 [55] RColorBrewer_1.1-3 ggridges_0.5.4 [57] Rcpp_1.0.10 plyr_1.8.8 [59] base64enc_0.1-3 sparseMatrixStats_1.10.0 [61] progress_1.2.2 zlibbioc_1.44.0 [63] RCurl_1.98-1.12 prettyunits_1.1.1 [65] viridis_0.6.3 pbapply_1.7-0 [67] GetoptLong_1.0.5 zoo_1.8-12 [69] cluster_2.1.4 variancePartition_1.28.0 [71] magrittr_2.0.3 data.table_1.14.8 [73] lmerTest_3.1-3 lmtest_0.9-40 [75] RANN_2.6.1 fitdistrplus_1.1-11 [77] hms_1.1.3 patchwork_1.1.2 [79] evaluate_0.21 xtable_1.8-4 [81] pbkrtest_0.5.2 RhpcBLASctl_0.23-42 [83] XML_3.99-0.9 gridExtra_2.3 [85] shape_1.4.6 compiler_4.2.0 [87] scater_1.26.0 KernSmooth_2.23-21 [89] crayon_1.5.2 minqa_1.2.5 [91] htmltools_0.5.5 tzdb_0.4.0 [93] geneplotter_1.76.0 DBI_1.1.3 [95] MASS_7.3-60 boot_1.3-28.1 [97] Matrix_1.5-4.1 cli_3.6.1 [99] rbibutils_2.2.13 metapod_1.6.0 [101] parallel_4.2.0 igraph_1.5.0 [103] pkgconfig_2.0.3 numDeriv_2016.8-1.1 [105] IRdisplay_1.1 plotly_4.10.0 [107] foreach_1.5.2 annotate_1.76.0 [109] vipor_0.4.5 dqrng_0.3.0 [111] blme_1.0-5 XVector_0.38.0 [113] digest_0.6.31 sctransform_0.3.5 [115] RcppAnnoy_0.0.20 tsne_0.1-3.1 [117] Biostrings_2.66.0 leiden_0.4.3 [119] uwot_0.1.14 edgeR_3.40.0 [121] DelayedMatrixStats_1.20.0 gtools_3.9.4 [123] rjson_0.2.21 nloptr_2.0.3 [125] lifecycle_1.0.3 nlme_3.1-157 [127] jsonlite_1.8.5 aod_1.3.2 [129] BiocNeighbors_1.16.0 viridisLite_0.4.2 [131] fansi_1.0.4 pillar_1.9.0 [133] lattice_0.21-8 KEGGREST_1.38.0 [135] fastmap_1.1.1 httr_1.4.6 [137] survival_3.5-5 glue_1.6.2 [139] png_0.1-8 iterators_1.0.14 [141] bluster_1.8.0 bit_4.0.5 [143] stringi_1.7.6 blob_1.2.4 [145] BiocSingular_1.14.0 DESeq2_1.38.0 [147] caTools_1.18.2 memoise_2.0.1 [149] IRkernel_1.3.2 irlba_2.3.5.1 [151] future.apply_1.11.0 ape_5.7-1